WO2018139548A1 - Milieu destiné à induire une différenciation de cellules souches en cellules mésodermiques et procédé destiné à produire des cellules mésodermiques - Google Patents

Milieu destiné à induire une différenciation de cellules souches en cellules mésodermiques et procédé destiné à produire des cellules mésodermiques Download PDF

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WO2018139548A1
WO2018139548A1 PCT/JP2018/002321 JP2018002321W WO2018139548A1 WO 2018139548 A1 WO2018139548 A1 WO 2018139548A1 JP 2018002321 W JP2018002321 W JP 2018002321W WO 2018139548 A1 WO2018139548 A1 WO 2018139548A1
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cells
stem cells
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明石　満
健 福本
信一郎 庄司
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国立大学法人大阪大学
協和発酵バイオ株式会社
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Priority to EP18745322.0A priority Critical patent/EP3575392A4/fr
Priority to US16/480,670 priority patent/US20210130785A1/en
Priority to JP2018564632A priority patent/JP7162537B2/ja
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present disclosure relates to a medium for inducing differentiation of stem cells into mesodermal cells, and a method for producing mesodermal cells from stem cells using the medium.
  • Non-Patent Documents 1 to 7 Yamanaka et al. Have developed a method for efficiently producing cardiomyocytes from stem cells by once forming EBs, dissociating and reaggregating the obtained EBs (Patent Document 1).
  • stem cells In inducing differentiation of stem cells into target cells, it is desirable to differentiate into target cells as efficiently as possible.
  • the conventional methods have a problem that it is necessary to examine the optimal EB formation period in advance and that it takes time necessary for EB formation. Therefore, there is a need for a rapid and simple method for inducing stem cell differentiation.
  • Rho-associated kinase Rho-associated coiled-coil forming kinase: ROCK
  • BMP bone morphogenetic factor
  • FGF fibroblast growth factor
  • activin activin
  • the present invention provides the following: [1] A medium for inducing differentiation into stem mesodermal cells, comprising a ROCK inhibitor, bone morphogenetic factor (BMP), fibroblast growth factor (FGF), and activin; [2] The medium according to [1], wherein the ROCK inhibitor is Y-27632.
  • BMP bone morphogenetic factor
  • FGF fibroblast growth factor
  • stem cells can be induced to differentiate into mesodermal cells with high efficiency. Furthermore, when suspension culture is used, a large amount of stem cells can be induced to differentiate into mesodermal cells without passing through the EB formation period.
  • FIG. 1 shows the culture conditions of Example 1 (Conditions 1 to 4).
  • FIG. 2 shows the transition of the viable cell density under conditions 1 to 4.
  • FIG. 3 shows the ratio of cTnT positive cells under conditions 1 to 4.
  • FIG. 4 shows the culture conditions of Example 2 (Conditions A to F).
  • FIG. 5 shows changes in the density of living cells under conditions A to F.
  • FIG. 6 shows the ratio of cTnT positive cells under conditions A, D and E.
  • the present invention is a medium for producing mesodermal cells from stem cells, comprising a ROCK inhibitor, BMP, activin, and FGF, a medium for inducing differentiation from stem cells to mesodermal cells (hereinafter referred to as “this”).
  • Inventive medium a method of producing mesodermal cells by culturing stem cells in the medium (hereinafter also referred to as“ method of the present invention ”), induction of differentiation of stem cells into mesodermal cells.
  • a composition for assisting hereinafter also referred to as “the composition of the present invention”
  • a cell group obtained by the method of the present invention hereinafter also referred to as “the cell group of the present invention”.
  • the medium of the present invention is a medium for inducing differentiation into stem cell mesodermal cells containing a ROCK inhibitor, BMP, FGF, and activin.
  • the medium of the present invention is a medium obtained by adding a component containing a ROCK inhibitor, BMP, FGF, and activin to a basal medium for stem cells.
  • stem cell refers to an immature cell having self-replication ability and differentiation / proliferation ability.
  • Stem cells have hierarchies, and upper undifferentiated stem cells have high self-replicating ability and high pluripotency capable of differentiating into various cell lineages. It is known that it can only differentiate into cell lineages.
  • the stem cell-derived species to which the medium of the present invention can be applied is not particularly limited, and examples thereof include rodents such as rats, mice, hamsters and guinea pigs, rabbits such as rabbits, and hoofs such as pigs, cows, goats and sheep. Eyes, cats such as dogs and cats, and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees.
  • Stem cells include pluripotent stem cells, multipotent stem cells, unipotent stem cells, etc., depending on their differentiation ability.
  • pluripotent stem cell means a stem cell that can be differentiated into all cells that can exist in a living body (that is, has pluripotency) and has proliferative ability.
  • pluripotent stem cells include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), cloned embryo-derived embryonic stem cells (ntES cells) obtained by nuclear transfer, sperm stem cells (GS cells) , Embryonic germ cells (EG cells), cultured fibroblasts, bone marrow stem cell-derived pluripotent cells (Muse cells), and the like, but are not limited thereto.
  • the pluripotent stem cell used may be prepared by a conventionally known method or may be a cell line that is generally available. Examples of ES cells include, but are not limited to, KhES1, KhES3, and the like.
  • the pluripotent stem cell used in the present invention is an iPS cell.
  • An iPS cell is a somatic cell-derived artificial stem cell having pluripotency and proliferative ability, which is produced by introducing a specific reprogramming factor into a somatic cell in the form of a nucleic acid or protein.
  • genes included in the reprogramming factor include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, Eras, Examples include ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1. These initialization factors may be used alone or in combination.
  • the iPS cells used in the present invention may be prepared by a conventionally known method or may be a cell line that is generally available.
  • Exemplary strains of human iPS cells include 253G1 (RIKEN Cell Bank No. HPS0002), 201B7 (RIKEN Cell Bank No. HPS0063), 409B2 (RIKEN Cell Bank No. HPS0076), 454E2 (RIKEN Cell Bank No. HPS0077), 606A1 (RIKEN) Cell Bank No. HPS0328), 610B1 (RIKEN Cell Bank No. HPS0331), 648A1 (RIKEN Cell Bank No.
  • HPS0360 MYH (Patent Literature 1), 427F1 (Patent Literature 1), 457C1 (Patent Literature 1), 604A1 (Patent Literature 1) ), HiPS-RIKEN-1A (RIKEN Cell Bank No. HPS0003), HiPS-RIKEN-2A (RIKEN Cell Bank No. HPS0009), HiPS-RIKEN-12A (RIKEN Cell Bank N) .HPS0029), Nips-B2 (RIKEN Cell Bank No.HPS0223) or the like include, but are not limited to.
  • Exemplary strains of iPS cells derived from non-human animals include iPS-MEF-Ng-20D-17, iPS-MEF-Ng-178B-5, iPS-MEF-Fb / Ng-440A-3, iPS-MEF -Ng-492B-4, iPS-Stm-FB / gfp-99-1, iPS-Stm-FB / gfp-99-3, iPS-Hep-FB / Ng / gfp-103C-1, iPS-L1, iPS -S1 and the like can be mentioned, but not limited thereto. Alternatively, disease specific iPS cells may be used.
  • diseases include blood system diseases, immune system diseases, endocrine diseases, metabolic diseases, visual diseases, cardiovascular diseases, respiratory diseases, skin / connective tissue diseases, bone / joint diseases, kidneys -Include, but are not limited to, urinary diseases, syndromes with changes in chromosomes or genes (see http://cell.brc.riken.jp/ja/hps/hps_dislistist_index).
  • examples of the above cell lines include the RIKEN BioResource Center (see http://cell.brc.riken.jp/en/) and the JCRB cell bank (http://cellbank.nibiohn.go.jp/). available from English /).
  • the iPS cells used in the present invention may be obtained by culturing on feeder cells. Or you may obtain by culture
  • multipotent stem cells examples include somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, and reproductive stem cells.
  • the multipotent stem cell is preferably a mesenchymal stem cell.
  • Mesenchymal stem cells are undifferentiated cells (somatic stem cells) present in adult bone marrow, adipose tissue, placental tissue, umbilical cord blood, dental pulp, etc., and proliferative and multipotent (especially bone cells, chondrocytes,
  • a broad term means a population of stem cells or progenitor cells having the ability to differentiate into muscle cells, tendon cells, fat cells, etc.).
  • mesenchymal stem cells include mesenchymal stem cells derived from human bone marrow (hMSC-BM, manufactured by Takara), mesenchymal stem cells derived from human umbilical cord matrix (hMSC-UC, manufactured by Takara), or mesenchymal cells derived from human adipose tissue.
  • System stem cells hMSC-AT, manufactured by Takara
  • any stem cell can be suitably used, but preferably a pluripotent stem cell or a mesenchymal stem cell, more preferably a pluripotent stem cell can be used.
  • the pluripotent stem cell is preferably an ES cell or iPS cell, more preferably an iPS cell, and particularly preferably a human iPS cell.
  • the “mesoderm cell” refers to a cell constituting a tissue derived from mesoderm.
  • Tissues derived from mesoderm include bone, cartilage, spleen, bone marrow, dentin, peritoneal epithelium, kidney, ureter, pleural epithelium, adrenal cortex, muscle (other than pupil sphincter and dilated pupil), ovary, uterus, testis , And connective tissue.
  • mesoderm cells include, but are not limited to, neuroglial cells, myocardial progenitor cells, cardiomyocytes, vascular endothelial progenitor cells, vascular endothelial cells, blood cells, and mesenchymal stem cells.
  • the “basal medium for stem cells” is not particularly limited as long as it can induce differentiation of stem cells into mesodermal cells by adding a ROCK inhibitor, BMP, FGF and activin.
  • the stem cell basal medium comprises one or more sugar (s), one or more inorganic salts (s), one or more amino acids (s), one or more vitamins (s), and one or more traces. Preferably it contains the component (s).
  • antibiotics such as kanamycin can be included as appropriate for use in drug sensitivity tests.
  • sugar (s) examples include monosaccharides such as glucose, lactose, mannose, fructose and galactose, and disaccharides such as sucrose, maltose and lactose.
  • monosaccharides such as glucose, lactose, mannose, fructose and galactose
  • disaccharides such as sucrose, maltose and lactose.
  • glucose is particularly preferable.
  • These saccharides can be added alone or in combination.
  • Examples of the inorganic salt (s) include calcium chloride, calcium nitrate, copper sulfate pentahydrate, iron (III) nitrate nonahydrate, iron (II) sulfate heptahydrate, magnesium chloride hexahydrate.
  • Any inorganic salt or a combination thereof can be used as long as it is a component that advantageously acts on differentiation induction of stem cells into mesodermal cells.
  • amino acid (s) examples include alanine, arginine, asparagine, aspartic acid, cystine, cysteine, glutamine, glycine, histidine, glutamic acid, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, Examples include tryptophan, tyrosine and valine, and L-amino acids are preferred.
  • the amino acid (s) may include derivatives such as derivatives, salts and hydrates thereof.
  • Examples of arginine derivatives include L-arginine hydrochloride and L-arginine monohydrochloride.
  • Examples of derivatives of aspartic acid include L-aspartic acid sodium salt monohydrate, L-aspartic acid monohydrate, potassium L-aspartate, and magnesium L-aspartate.
  • Examples of cysteine derivatives include L-cysteine dihydrochloride and L-cysteine hydrochloride monohydrate.
  • Examples of lysine derivatives include L-lysine hydrochloride.
  • Examples of the derivative of glutamic acid include L-glutamic acid monosodium salt and the like.
  • As a derivative of asparagine for example, L-asparagine monohydrate and the like can be mentioned.
  • Examples of the derivative of tyrosine include L-tyrosine disodium dihydrate and the like.
  • examples of histidine derivatives include histidine hydrochloride and histidine hydrochloride monohydrate.
  • Examples of lysine derivatives include L-lysine hydrochloride.
  • vitamin (s) examples include ascorbic acid, biotin, choline, folic acid, inositol, niacin, pantothenic acid, pyridoxine, riboflavin, thiamine, vitamin B12, and paraaminobenzoic acid (PABA).
  • ascorbic acid is preferably added.
  • the vitamin (s) can include derivatives such as derivatives, salts and hydrates thereof.
  • Examples of derivatives of ascorbic acid include ascorbic acid 2-phosphate ester (Ascorbic acid 2-phosphate), magnesium ascorbate phosphate, sodium ascorbate sulfate, ascorbyl aminopropyl phosphate, and sodium ascorbate phosphate. It is done.
  • Examples of the derivative of choline include choline chloride.
  • Examples of niacin derivatives include nicotinic acid, nicotinamide and nicotinic alcohol.
  • Examples of the derivative of pantothenic acid include calcium pantothenate, sodium pantothenate, panthenol and the like.
  • Examples of the derivative of pyridoxine include pyridoxine hydrochloride, pyridoxal hydrochloride, pyridoxal phosphate, and pyridoxamine.
  • Derivatives of thiamine include, for example, thiamine hydrochloride, thiamine nitrate, bisthiamine nitrate, thiamine dicetyl sulfate ester salt, fursultiamine hydrochloride, octothiamine and benfotiamine.
  • the minor component (s) is preferably a component that advantageously acts on differentiation induction of stem cells into mesodermal cells.
  • the trace component (s) include components used as usual medium components such as glutathione, hypoxanthine, lipoic acid, linolenic acid, phenol red, putrescine, pyruvic acid, thymidine, and NaHCO 3 .
  • the trace component (s) can include derivatives such as derivatives, salts and hydrates thereof. Examples of the derivative include putrescine dihydrochloride.
  • basal medium for stem cells a basal medium known to those skilled in the art can be used.
  • commercially available culture media such as StemPro (registered trademark) -34 (Thermo Fisher Scientific), mESF Basal Medium (Wako Pure Chemicals), etc.
  • MEM Minimum Essential Medium
  • BME Basic Medium Eagle
  • DMEM Dulbecco's Modified Eagle Medium
  • EMEM Eagle's minimalEsdM (Glas-gow's MEM)
  • F12 Ham's F12 Mediu
  • DMEM / F12 medium in which DMEM and F12 medium are mixed at 1: 1)
  • RPMI1640 RD
  • BMC-3 Brinster's BMOC-3 Medium
  • CMRL-1066 L-15 medium (Leibovitz's L -15 medium)
  • McCoy's 5A Media 199, MEM ⁇ Media
  • MCDB105 MCDB131
  • MCDB153 MCDB201
  • Williams' medium E, and ESF Williams' medium E, and ESF.
  • the basal medium for stem cells may be KnockOut (trademark) Serum Replacement (KSR) (Thermo Fisher Scientific), StemSure Serum Replacement (SSR) (Wako Pure Chemicals, etc.) Serum-free supplements such as B27 Replacement (Thermo Fisher Scientific) may be added.
  • KSR Serum Replacement
  • SSR StemSure Serum Replacement
  • B27 Replacement Thermo Fisher Scientific
  • Examples of such a medium include, for example, a medium in which KSR is added to MEM, BME, DMEM, EMEM, IMDM, DMEM / F12, and RPMI1640, and B27 Replacement to MEM, BME, DMEM, EMEM, IMDM, DMEM / F12, and RPMI1640.
  • StemPro (registered trademark) -34 preferably, MEM, BME, DMEM, EMEM, IMDM, DMEM / F12 and RPMI1640 to which B27 Replacement is added, and StemPro (registered trademark) -34, More preferably, a medium in which B27 Replacement is added to RPMI1640, and StemPro (registered trademark) -34, most preferably StemPro (registered trademark) -34 are listed. Rukoto can.
  • additives usually used for cell culture may be further added.
  • additives include, but are not limited to, L-ascorbic acid 2-trisodium phosphate, L-glutamine, 1-thioglycerol and the like.
  • the type and amount of the medium used can be appropriately selected by those skilled in the art according to the type of cells to be cultured.
  • ROCK inhibitors used in the present invention include (R)-(+)-trans-N- (4-pyrylyl) -4- (1-aminoethyl) -cyclohexanecarbamide, 2HCl, H 2 O (Y-27632 ), 1- (5-isoquinolinesulfonyl) piperazine hydrochloride (HA100), 1- (5-isoquinolinesulfonyl) homopiperazine dihydrochloride (Fasudil / HA-1077), (S)-(+)-2-methyl- 4-glycyl-1- (4-methylisoquinolinyl-5-sulfonyl) homopiperidine dihydrochloride (H-1152), 1- (5-isoquinolinesulfonyl) -2-methylpiperazine (H-7), 1 -(5-isoquinolinesulfonyl) -3-methylpiperazine (isoH-7), N-2- (methyla C) ethyl-5-isoquinolinesulf
  • the concentration of the ROCK inhibitor used in the present invention is about 0.2 to about 100 ⁇ M, for example, about 1 to about 75 ⁇ M, about 2 to about 50 ⁇ M, or about 5 to about 20 ⁇ M. In an exemplary embodiment, the concentration of ROCK inhibitor used is about 10 ⁇ M.
  • BMP used in the present invention examples include, but are not limited to, BMP2, BMP4, BMP6, and BMP8.
  • the BMP used is BMP4.
  • the concentration of BMP used in the present invention is about 0.5 to about 500 ng / mL, such as about 1 to about 100 ng / mL, about 2 to about 50 ng / mL, or about 5 to about 20 ng / mL. In an exemplary embodiment, the concentration of BMP used is about 10 ng / mL.
  • FGF used in the present invention examples include FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, and FGFv9. It is not limited to.
  • the FGF used is FGF-2.
  • the concentration of FGF used in the present invention is about 0.1 to about 250 ng / mL, such as about 0.5 to about 50 ng / mL, about 1 to about 25 ng / mL, or about 2.5 to about 10 ng / mL. It is. In an exemplary embodiment, the concentration of FGF used is about 5 ng / mL.
  • activins used in the present invention include, but are not limited to, activin A, activin B, and activin AB.
  • the activin used is activin A.
  • the concentration of activin used in the present invention is about 0.12 to about 300 ng / mL, such as about 0.6 to about 60 ng / mL, about 1.2 to about 30 ng / mL, or about 3 to about 12 ng / mL. It is. In an exemplary embodiment, the concentration of activin used is about 6 ng / mL.
  • the method of the present invention is a method for producing mesodermal cells from stem cells, comprising culturing the stem cells in the medium of the present invention described in 1 above.
  • the culture conditions such as culture temperature and culture time in the method of the present invention can be appropriately selected by those skilled in the art depending on the type of cells to be cultured.
  • the pluripotent stem cells are cultured in the medium of the present invention at 34 to 40 ° C./2 to 8% CO 2 for 12 hours to 7 days. Incubate.
  • the cells are cultured at 35 to 39 ° C./3 to 7% CO 2 for 1 to 4 days.
  • the medium of the present invention containing the above four factors (ROCK inhibitor, BMP, FGF, and activin) at 36 to 38 ° C./4 to 6% CO 2 for 6 to 42 hours, preferably 12 After culturing for a time to 36 hours, more preferably 18 to 30 hours, in a medium containing 3 factors (BMP, FGF, and activin) excluding the ROCK inhibitor at 36 to 38 ° C./4 to 6% CO 2
  • the culture is performed for 12 hours to 4 days, preferably 1 to 3 days, more preferably 36 to 60 hours, and most preferably 42 to 54 hours.
  • the pluripotent stem cells are cultured in the medium of the present invention at 34 to 40 ° C./2 to 8% CO 2 for 12 hours to 7 days. To do. Preferably, the cells are cultured at 35 to 39 ° C./3 to 7% CO 2 for 1 to 4 days, more preferably 2 to 4 days.
  • the types, concentrations, etc. of the ROCK inhibitor, BMP, FGF, and activin contained in the medium are as described above.
  • the ROCK inhibitor used is Y-27632
  • BMP is BMP4
  • FGF is FGF-2
  • activin activin A.
  • adhesion culture or suspension culture can be used, and as a preferred form, suspension culture can be used.
  • Suspension culture can be performed using a means, method, or apparatus that can keep cells floating.
  • incubators with stirring blades such as single-use bioreactors (Biot Co., Ltd.), single-use bioreactors (Thermo Fisher), single-use bioreactors (Sartorius Stedium), single-use bioreactors (GE Healthcare Life Sciences) It can be implemented by using.
  • the type of the incubator used and the stirring speed can be appropriately selected by those skilled in the art depending on the type of cells to be cultured. Examples of the stirring speed include, but are not limited to, 0 to 100 rpm, 20 to 80 rpm, or 45 to 65 rpm.
  • the mesoderm cells are as described above.
  • inducing differentiation of stem cells into mesodermal cells a method well known to those skilled in the art is used to induce differentiation of mesoderm-derived tissue from mesoderm after culturing the stem cells in the medium of the present invention. be able to.
  • differentiation medium suitable for differentiation into a desired mesoderm-derived tissue differentiation into a desired mesoderm cell can be induced (Nathan J Palpant. Al. Nature Protocols 2016 Dec; 12 (1): 15-31, Cythia A. Batchelder et.al.2009 Jul; 78 (1): 45-56 etc.).
  • the stem cells may be induced to differentiate into mesenchymal stem cells, and the obtained mesenchymal stem cells may be differentiated into mesodermal cells.
  • Methods for inducing differentiation of mesenchymal stem cells into mesodermal cells are well known to those skilled in the art.
  • mesenchymal stem cells can be induced to differentiate into mesodermal cells by culturing mesenchymal stem cells in a differentiation medium suitable for differentiation into desired cells (Nishiyama et al. 2007 Aug; 25 (8): 2017-2024 etc.).
  • the present invention provides a method for producing myocardial progenitor cells or cardiomyocytes from stem cells.
  • the method is a method comprising (1) culturing stem cells in the above-described medium of the present invention, and (2) culturing in a medium containing a Wnt inhibitor. Step (2) may be followed by (3) culturing in a medium containing vascular endothelial growth factor (VEGF) and FGF.
  • VEGF vascular endothelial growth factor
  • the above-mentioned step (2) is a step of culturing in a medium containing IWR-1 and IWP-2, and the above-mentioned step (3) is cultivated in a medium containing VEGF and FGF-2. It is a stage.
  • a Wnt inhibitor refers to a substance that inhibits the Wnt signal transduction pathway.
  • IWR-1 (4-[(3aR, 4S, 7R, 7aS) -1,3,3a, 4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H -Isoindol-2-yl] -N-8-quinolinyl-benzamide
  • IWP-2 N- (6-Methyl-2-benzothiozyl) -2-[(3,4,6,7-tetrahydr-4--4-oxo) -3-phenylthieno [3,2-d] pyrimidin-2-yl) thio] -acetamide
  • WntC59 (4- (2-methy1-4-pyridinyl) -N- (4- (3-pyridinyl) phenyl)- benzeneacetamide
  • IWP4 N- (6-met yl-2-benzothiazolyl) -2-[[3,4,6,7-tetrahydro-3
  • the concentration of IWR-1 used is, for example, about 0.4 to about 40 ⁇ M, about 0.8 to about 20 ⁇ M, or about 2 to about 8 ⁇ M. In an exemplary embodiment, the concentration of IWR-1 used is about 4 ⁇ M.
  • the concentration of IWP-2 used is, for example, from about 1 to about 100 ⁇ M, from about 2 to about 50 ⁇ M, or from about 5 to about 20 ⁇ M. In an exemplary embodiment, the concentration of IWP-2 used is about 10 ⁇ M.
  • the concentration of VEGF used is about 0.1 to about 100 ng / mL, such as about 0.5 to about 50 ng / mL, about 1 to about 25 ng / mL, or about 2.5 to about 10 ng / mL. In an exemplary embodiment, the concentration of VEGF used is about 5 ng / mL.
  • the concentration of FGF-2 used is about 0.1 to about 250 ng / mL, such as about 1 to about 100 ng / mL, about 2 to about 50 ng / mL, or about 5 to About 20 ng / mL. Specific examples of FGF include FGF 1 described above. In an exemplary embodiment, FGF-2 is used as FGF, and the concentration of FGF-2 used is about 10 ng / mL.
  • step (2) may be culturing for 1 to 7 days at 35 to 39 ° C./3 to 7% CO 2 .
  • the step (3) may be culturing for 1 to 20 days at 35 to 42 ° C./3 to 7% CO 2 . More preferably, step (2) is culturing for 2 to 4 days at 36-38 ° C./4-6% CO 2 and step (3) is 7 for 36-40 ° C./4-6% CO 2. Incubation for ⁇ 15 days.
  • composition of the present invention is a composition for assisting inducing differentiation of stem cells into mesodermal cells, comprising a ROCK inhibitor, BMP, activin, and FGF.
  • the types, concentrations, and the like of the ROCK inhibitor, BMP, activin, and FGF contained in the above composition are as described above.
  • the ROCK inhibitor used is Y-27632
  • BMP is BMP4
  • activin is activin A
  • FGF is FGF-2.
  • the composition may be a liquid composition or a powdered composition such as a lyophilized product.
  • the cell group of the present invention is a cell group obtained by the above-described method of the present invention, and about 90% or more of mesodermal cells obtained by differentiation induction. In the cell population, about 90% or more, such as about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more Are mesodermal cells.
  • iPS cell line 253G1 which is a pluripotent stem cell
  • MEF cells CF-1 MEF
  • Maintenance culture was performed in a medium for iPS cells.
  • KnockOut TM DMEM / F12 was added to KnockOut TM Serum Replacement (KSR) (final concentration 20%), 0.1 mM MEM non-essential amino acid solution, 1 mM L-glutamine, 0.1 mM ⁇ -Mercaptoethanol with 4 ng / mL FGF-2 added was used.
  • KSR KnockOut TM Serum Replacement
  • Viable cell counting Cells were detached using Accutase or AccuMax (Innovative Cell Technologies). After staining with trypan blue, viable cells were counted using a hemocytometer.
  • Alexa Floor 488 goat anti-mouse IgG1 (1: 100) was then added as a secondary antibody.
  • Cells were washed twice with Perm / Wash TM buffer. The cells were washed once with 2% FBS in PBS, resuspended, and samples were measured and analyzed using Cell Sorter SH800 (Sony).
  • Example 1 Effect on the induction of cardiomyocyte differentiation by the presence or absence of EB formation StemPro (registered trademark) -34 SFM (Thermo Fisher Scientific) to L-ascorbic acid 2-trisodium phosphate (final concentration 50 ⁇ g / mL), 2 mM L A medium supplemented with glutamine and 400 ⁇ M 1-thioglycerol was used (hereinafter referred to as “basal medium”). According to the culture conditions outlined in FIG. 1 (conditions 1 to 4), iPS cells were induced to differentiate into cardiomyocytes.
  • the iPS cells cultured in advance were detached and collected by Accutase treatment, and then centrifuged to remove the supernatant.
  • the collected iPS cells were suspended in a medium.
  • iPS cells were suspended in a basal medium containing 10 ⁇ M Y-27632.
  • iPS cells were suspended in a basal medium containing 10 ⁇ M Y-27632, 10 ng / mL BMP-4, 5 ng / mL FGF-2, and 6 ng / mL activin A.
  • Suspended iPS cells were seeded into Single use bio-reactor for 30 mL (Biot Co., Ltd.) at 4.0 ⁇ 10 6 cells / reactor.
  • the culture was performed at a stirring speed of 55 rpm and 37 ° C./5% CO 2 .
  • Conditions 1 to 3 were cultured in a basal medium containing 10 ⁇ M Y-27632 for one day (condition 1: Day-3 to -2, condition 2: Day-2 to -1, condition 3: Day-1 to 0). . After culturing, conditions 1 and 2 were further cultured in a basal medium for 2 days and 1 day, respectively (condition 1: Day-2 to 0, condition 2: Day-1 to 0).
  • the cells were cultured for 3 days in a basal medium containing 10 ng / mL BMP-4, 5 ng / mL FGF-2, and 6 ng / mL activin A (conditions 1-3: Day 0 to 3).
  • condition 4 After stirring for 1 day in basal medium containing 10 ⁇ M Y-27632, 10 ng / mL BMP-4, 5 ng / mL FGF-2, and 6 ng / mL activin A (condition 4: Day 0 to 1) The cells were cultured for 2 days in a basal medium containing 10 ng / mL BMP-4, 5 ng / mL FGF-2, and 6 ng / mL activin A (condition 4: Day 1 to 3). After culturing under each condition, the cell mass was recovered from the reactor and transferred to a centrifuge tube.
  • the cell mass was washed with a basal medium, then resuspended in a basal medium containing 4 ⁇ M IWR-1 and 10 ⁇ M IWP-2, and cultured for 3 days (conditions 1 to 4: Day 3 to 6). Thereafter, the medium was replaced with a basal medium containing 5 ng / mL VEGF and 10 ng / mL FGF-2 and cultured for a maximum of 10 days (Condition 1 to 4: Day 6 to). In culture with these media, the media was changed every two days.
  • the maximum value of the viable cell density under each condition was as shown in the following table (FIG. 2).
  • the maximum value of the cardiomyocyte marker (Cardiac Troponin T: cTnT) in each condition was as shown in the following table (FIG. 3).
  • condition 4 without EB formation period had more viable cells and more cTnT positive cells than conditions 1 to 3 with EB formation period.
  • Example 2 Effect on differentiation induction efficiency due to difference in factors added to basal medium Factors shown in the following table were added to the basal medium.
  • the iPS cells cultured in advance were detached and collected by Accutase treatment, and then centrifuged to remove the supernatant.
  • the collected iPS cells were suspended in each of the media under conditions A to F, and then seeded into 30 mL of single use bio-reactor for at 5.07 ⁇ 10 6 cells / reactor.
  • the culture was performed at a stirring speed of 55 rpm and 37 ° C./5% CO 2 .
  • the culture procedure for each condition is outlined in FIG. For conditions A and C to E, after culturing for one day in the medium of each condition (conditions A and C to E: Day 0 to 1), each medium was replaced with a medium except 10 ⁇ M Y-27632.
  • condition A and C to E Day 1 to 3
  • condition B and F after culturing for 1 day under each condition (conditions B and F: Day 0 to 1), the medium was changed and further cultured for 2 days under the same conditions (conditions B and F: Day 1 to 3).
  • the cell mass was recovered from the reactor and transferred to a centrifuge tube. After washing the cell mass with basal medium, the cells were resuspended in basal medium containing 4 ⁇ M IWR-1 and 10 ⁇ M IWP-2, and cultured for 3 days (conditions AF: Day 3-6). Thereafter, the medium was replaced with a basal medium containing 5 ng / mL VEGF and 10 ng / mL FGF-2 and cultured for a maximum of 10 days (conditions A to F: Day 6 to).
  • the maximum value of the viable cell density under each condition is shown in the following table (FIG. 5). * : The cell died during differentiation induction. In addition, the maximum value of the cardiomyocyte marker (Cardiac Troponin T: cTnT) under each condition in which the cells survived was as shown in the following table (FIG. 6).
  • Example 3 Measurement of cell pulsation A culture solution (2 ml) containing a myocardial cell mass obtained by inducing differentiation under the condition A in Example 2 was transferred to a 12 well plate MICROPPLATE with Lid (IWAKI), and Leica It observed with DMi1 inverted microscope (Leica). As a result, it was confirmed that each myocardial cell mass was autonomously beating at a constant cycle.
  • Example 4 Measurement of Ca + imaging Using Calcium Kit-Fluo 4 (Dojindo Laboratories), Ca + imaging was performed on cell masses and cells adhered on the substrate. Differentiation was induced for 16 days under the condition A of Example 2 to obtain a cell mass. The obtained cell mass was washed with PBS, suspended in Loading Buffer, and transferred to MATUNAMI GLASS BOTTOM DISH Hydro 35mm dish (Matsunami Glass Industrial Co., Ltd.). Then, it incubated at 37 degreeC for 1 hour. After the incubation, the loading buffer was removed and washed with PBS. The obtained cell mass was transferred to Recording Medium, and fluorescence observation was performed with ECLIPSE Ts2 (Nikon).
  • a cell mass obtained by performing differentiation induction for 16 days under the condition A in Example 2 was treated with AccuMax (Innovation cell technologies), and the cell mass was detached.
  • the detached cell mass was suspended in DMEM + 10% FBS, and the suspension was passed through a 40 ⁇ m strainer (FALCON).
  • Ca + imaging of the cells adhered on the substrate was performed in the same manner as described above. As a result, it was possible to confirm that the Ca + concentration increased in synchronism with the pulsation of the cell mass and the adhered cells.
  • Example 5 Expression of myocardial marker A cell mass obtained by inducing differentiation for 16 days under the condition A in Example 2 was treated with AccuMax (Innovation cell technologies), and the cell mass was detached. The suspension was suspended in DMEM + 10% FBS, and the suspension was passed through a 40 ⁇ m strainer (FALCON). Cells diluted with DMEM + 10% FBS so as to be 4 ⁇ 10 4 cells / well are seeded in 24 well plate MICROPLATE with Lid (IWAKI) previously coated with fibronectin (5 ⁇ g / cm 2 , Sigma), and in DMEM + 10% FBS For 6 days.
  • the culture supernatant was removed, and after washing 3 times with PBS, cells were fixed with Fixation / Permeabilization solution (4 ° C., 60 minutes or overnight). After washing 3 times with PBS, blocking with PBS + 3% FBS was performed at room temperature for 30 minutes. The solution was discarded and replaced with PBS + 1% FBS containing various antibodies (4 ° C., 60 minutes or overnight). The solution was discarded and replaced with 0.1% FBS / PBST, and left to stand for 5 minutes for washing (stationary washing). This washing operation was performed three times. After removing the washing solution, various secondary antibodies were added (room temperature, 60 minutes). After performing static washing with PBS for 5 minutes three times, it was treated with DAPI (Life technologies) (room temperature, 15 minutes).
  • Example 6 Measurement of extracellular potential A cell mass obtained by inducing differentiation for 14 days under the condition A in Example 2 was treated with AccuMax (Innovation cell technologies), and the cell mass was detached. The suspension was suspended in DMEM + 10% FBS, and the suspension was passed through a 40 ⁇ m strainer (FALCON). In advance, a frame was made of silicon so as to surround the electrode of multi-electrode dish MED-P530A (Alphamed Scientific Co., Ltd.), and then the inside was coated with fibronectin (about 5 ⁇ g / cm 2 , Sigma). 1 ⁇ 10 5 cells were seeded in a frame and allowed to stand for 1 hour in a 37 ° C., 5% CO 2 incubator.
  • FALCON 40 ⁇ m strainer
  • the medium was replenished and cultured for 4 days.
  • the extracellular potential was measured with MED64 (Alpha Med Scientific Co., Ltd.). As a result, it was confirmed that depolarization and repolarization occurred autonomously in the induced cardiomyocytes.
  • iPS cells were suspended in culture using a medium supplemented with the above four factors (ROCK inhibitor, BMP4, FGF-2, and activin A), and without undergoing preparation steps such as EB formation. It was shown that mesoderm cells can be induced and the obtained mesoderm cells can be induced into cardiomyocytes. It was also demonstrated that cardiomyocytes can be induced with high survival rate and high efficiency. Furthermore, it was confirmed that the induced cardiomyocytes beat autonomously at a constant cycle. Furthermore, it was confirmed that depolarization and repolarization occur autonomously in induced cardiomyocytes. That is, it was shown that cardiomyocytes can be induced quickly and easily with high efficiency by using the method of the present invention. This method is also suitable for mass culture of cells when suspension culture is used.
  • Example 7 Cardiomyocyte differentiation using feeder-free cultured iPS cells Stem Cell and Culture Method
  • iPS cell line 253G1 which is a pluripotent stem cell
  • the feeder-free iPS cell culture was performed under the following conditions. Incubator was coated with iMatrix® 511 at 0.5 ng / cm 2 . StemFit (registered trademark) AK02N was used as a medium for iPS cells.
  • the cultured cells were washed with PBS and then detached with 0.5 ⁇ TrypLE (trademark). Thereafter, the supernatant was removed by centrifugation.
  • the collected iPS cells were suspended in the medium of Condition A of Example 2 and then seeded into 30 mL of Single use bio-reactor for 5.0 ⁇ 10 6 cells / reactor.
  • the culture was performed at a stirring speed of 55 rpm and 37 ° C./5% CO 2 .
  • Culturing was performed in the medium of Condition A of Example 2 for 3 days. Thereafter, the cell mass was recovered from the reactor and transferred to a centrifuge tube.
  • the supernatant was removed, the cell mass was washed once with basal medium, resuspended in basal medium containing 4 ⁇ M IWR-1 and 10 ⁇ M IWP-2, and cultured again in the reactor for 3 days. After the culture, the medium was replaced with a basal medium containing 5 ng / mL VEGF and 10 ng / mL FGF-2, and the cells were cultured for a maximum of 15 days. During the culture, the medium was changed every two days.
  • the cardiomyocyte marker (Cardiac Troponin T: cTnT) in each differentiation induction period was as shown in the following table.
  • mesodermal cells can be induced from iPS cells cultured in a feeder-free state using the medium and method of the present invention without undergoing preparatory steps such as EB formation. It was shown that the obtained mesodermal cells can be induced into cardiomyocytes with high efficiency.
  • the present invention it is possible to induce differentiation of stem cells into mesodermal cells such as cardiomyocytes quickly and easily with high efficiency.
  • This method is also suitable for mass culture of mesodermal cells when suspension culture is used. Therefore, the present invention is useful in the field of regenerative medicine. The present invention is also useful in pharmaceutical development.

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Abstract

La présente invention concerne un milieu destiné à induire la différenciation de cellules souches en cellules mésodermiques, le milieu comprenant un inhibiteur de ROCK, une protéine morphogénétique osseuse (PMO), un facteur de croissance des fibroblastes (FCF) et de l'activine. La présente invention concerne également un procédé destiné à produire des cellules mésodermiques à partir de cellules souches à l'aide du milieu, et un procédé destiné à produire des cellules progénitrices cardiaques ou des myocardiocytes à partir de cellules souches à l'aide du milieu. La présente invention concerne en outre une composition destinée à aider à induire la différenciation de cellules souches en cellules mésodermiques, la composition comprenant un inhibiteur de ROCK, une PMO, un FCF et de l'activine. La présente invention concerne de surcroît des groupes de cellules obtenus par les procédés ci-dessus.
PCT/JP2018/002321 2017-01-26 2018-01-25 Milieu destiné à induire une différenciation de cellules souches en cellules mésodermiques et procédé destiné à produire des cellules mésodermiques WO2018139548A1 (fr)

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US16/480,670 US20210130785A1 (en) 2017-01-26 2018-01-25 Medium for inducing differentiation of stem cells into mesodermal cells and method for producing mesodermal cells
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JP7520813B2 (ja) 2019-03-29 2024-07-23 株式会社カネカ 多能性幹細胞の製造方法
WO2022181776A1 (fr) * 2021-02-25 2022-09-01 学校法人関西医科大学 Procédé d'induction de la différenciation de cellules du mésothélium péritonéal à partir de cellules souches pluripotentes
WO2023210661A1 (fr) * 2022-04-25 2023-11-02 国立大学法人金沢大学 COMPOSITION POUR RÉGULER L'ACTIVATION DES CELLULES CAR-T, LA PROLIFÉRATION DES CELLULES SOUCHES HÉMATOPOÏÉTIQUES ET LA DIFFÉRENCIATION DES CELLULES iPS, ET SON UTILISATION

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JPWO2018139548A1 (ja) 2019-11-14

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